CN108727584B

CN108727584B - Anti-cancer conjugates

Info

Publication number: CN108727584B
Application number: CN201710263126.1A
Authority: CN
Inventors: 袁建栋; 黄仰青; 宋云松
Original assignee: Borui Biomedical Suzhou Co ltd
Current assignee: Borui Biomedical Suzhou Co ltd
Priority date: 2017-04-21
Filing date: 2017-04-21
Publication date: 2021-01-05
Anticipated expiration: 2037-04-21
Also published as: CN108727584A

Abstract

The invention discloses a structure withA multi-branched drug conjugate of formula (i) or a pharmaceutically acceptable salt thereof:r is an organic center, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, q is any integer between 3 and 8, wherein L is:

Description

Anti-cancer conjugates

Technical Field

The invention relates to a multi-arm polymer modified targeting anticancer conjugate, in particular to a conjugate formed by connecting a targeting molecule and an anticancer drug through a multi-arm polymer.

Background

Over the years, various methods have been proposed for improving the stability and delivery of bioactive agents. Challenges associated with the formulation and delivery of pharmaceutical agents may include: poor water solubility, toxicity, low bioavailability, instability, and rapid in vivo degradation of the pharmaceutical agent. Although many approaches have been devised to improve the delivery of pharmaceutical agents, none of the individual approaches have had their drawbacks. For example, drug delivery methods commonly employed aim to solve or at least ameliorate one or more of the following problems, including drug encapsulation, such as in a liposome, polymer matrix, or unimolecular micelle, covalent attachment to a water soluble polymer such as polyethylene glycol, use of gene targeting agents, salt structures, and the like.

WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a drug nktr 102 in the third clinical stage, which is mainly used for metastatic breast cancer and developed by Nektar Therapeutics. The drug is a water-soluble multi-branched polymer prodrug to improve the load of the drug, and has the following structure:

the compound is connected with irinotecan by multi-arm PEG, so as to improve water solubility, increase drug loading and reduce side effects under the condition of unchanged anticancer effect. However, the drug still has the disadvantages of poor targeting, incapability of acting on specific cancer cells, capability of killing the cancer cells and influencing the performance of normal cells, and high incidence of adverse reaction.

Disclosure of Invention

The invention discloses a brand-new targeting multi-branched-chain drug conjugate, which has three or more branched chains and can be expressed as the following formula:

r is an organic center, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, q is any integer between 3 and 8, wherein L is:

the symbol "#" represents the point of attachment of the multivalent linker L to the targeting molecule T, "#" represents the point of attachment of the multivalent linker L to the active agent D, "%" represents the point of attachment of the multivalent linker L to POLY, wherein L is any integer between 2 and 20, and m and n are each any integer between 0 and 10;

d is camptothecin drug shown as formula (II):

R₁-R₅independently of one another, from the following groups: hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxy, cyanoNitro, azido, amide, hydrazine, amine, substituted amine, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, alkylsulfonyloxy; r₆Is H OR OR₈，R₈Is alkyl, alkenyl, cycloalkyl, haloalkyl or hydroxyalkyl; r₇Is hydroxyl, amino, or thiol.

POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, and the four together form the "branches" of the multi-branched drug conjugate. Each of the branches and the other branches of the multi-branched drug conjugate are independent of each other. Each branch emanates from an organic center "R". However, in general, each branch of the conjugate is the same.

Each of the variable moieties in structural formula (I) will now be described in detail.

Organic center, "R"

In formula (I), "R" is an organic core group of 1 to 100 atoms. Preferably, R contains from 3 to 50 atoms, and more preferably, R contains from about 3 to 30 atoms. R may be a core of all carbon atoms, and may optionally contain 1 or more heteroatoms, e.g., O, S, N, P, etc., depending on the particular central molecule used. R may be linear, branched or cyclic, giving rise to at least 3 independent polymer branches. In formula (I), "q" corresponds to the number of polymer branches emanating from "R".

The organic center "R" is derived from a molecule that provides a number of polymer attachment sites, approximately equal to the number of polymer branches. More preferably, the main central formula of the multi-chain polymer structure is at least the residue of a polyol, polysulfide or polyamine compound having 3 and more than 3 hydroxyl, thio or amino groups suitable as polymer branches. A "polyol" is a molecule consisting of a plurality (greater than 2) of available hydroxyl groups. A "polysulfide" is a molecule that consists of a plurality (greater than 2) of available thio groups. A "polyamine compound" is a molecule consisting of a plurality (greater than 2) of available amine groups. Depending on the number of polymeric branches, the precursor of the polyol, polyamine compound or polysulfide (prior to covalent bonding of POLY) typically comprises from 3 to 25 hydroxyl, thio or amino groups, preferably from 3 to 10 hydroxyl, thio or amino groups, and most preferably from 3 to about 8 (e.g., 3, 4, 5, 6, 7 or 8) hydroxyl, thio or amino groups suitable for covalent bonding with POLY.

The precursor of the polyol or polyamine centre typically has a formula R- (OH) p or R- (NH) prior to reaction with the polymer₂) p is the same as the formula (I). In formula (I), the values of p and q correspond, since each functional group in the parent organic molecule typically has an-OH and-NH group₂If the positions are susceptible or susceptible to reaction, they are covalently bonded to POLY of the polymer branches. In formula (I), the hydroxyl groups of the polyol of the R parent have been converted to a polymer branch after attachment to POLY, and R is depicted as the residue after attachment. For example, if the organic center molecule is derived from pentaerythritol, the precursor of the polyol has the formula C (CH)₂OH)₄The organic central group R is represented by:

illustrative preferred polyhydroxy compounds as the polymer center include aliphatic polyhydroxy compounds containing 1 to 10 carbon atoms and 1 to 10 hydroxyl groups, for example, ethylene glycol, alkylene glycols, hydrocarbon glycols, alkylene hydrocarbon glycols, hydrocarbon cycloalkyl glycols, 1, 5-decalin glycols, 4, 8-bis (hydroxymethyl) tricyclodecane, cycloalkylene glycols, dihydroxyalkanes, trihydroxyalkanes, tetrahydroxyalkanes, and the like. Cycloaliphatic polyols include straight or closed ring sugars and sugar alcohols such as mannitol, sorbitol, cellosolve, xylitol, bromitol, threitol, arabitol, erythritol, hexitol, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagatose, pyranoside, sucrose, lactose, maltose and the like. Aromatic polyols such as phenylphosphonium diol, hydrocarbyl phenylphosphonium diol, pyrogallol, fluoroglycinol, 1,2, 4-benzenetriol, resorcinol, hydrocarbyl resorcinol, dihydrocarbyl resorcinol, orcinol monohydrate, olivetol, hydroquinone, hydrocarbyl hydroquinone, phenyl hydroquinone, and the like may also be used. Other polyol centers that may be employed may include crown ethers, cyclodextrins, dextrins, or other carbohydrates.

In the structural formula (I), q is the number of polymer branches connected to corresponding R, and the specific number can be 3-20. Typically, specific numbers for "q" are 3, 4, 5, 6, 7, 8. Specifically, three, four, five, six, seven, eight polymer branches are emitted centering on "R".

In some embodiments, "R" has three polymer branches, "R" is preferably:

in some embodiments, "R" has four polymer branches, "R" is preferably:

in some embodiments, "R" has six polymer branches, "R" is preferably:

in some embodiments, "R" has eight polymer branches, "R" is preferably:

polymer, "POLY"

In formula (I), "POLY" is a polymer and the POLY in each polymer branch is independently selected, preferably each polymer is the same polymer, more preferably each polymer branch in formula (I) is the same. Preferred polymers are water soluble, any water soluble polymer may be used to form the conjugates of the invention, and any polymer referred to herein may be of any geometry or form. Representative polymers include, but are not limited to: polyethylene glycol, polypropylene glycol, poly (vinylpyrrolidone), poly (hydroxyalkyl methacrylate amine), poly (hydroxyalkyl methacrylate), poly (saccharide), poly (± -hydroxy acid), poly (acrylic acid), poly (vinyl acetic acid), polyphosphazine, polyoxazoline, poly (N-acryloylmorpholine), and the like.

In typical compounds, "POLY" is polyethylene glycol (PEG) and can be in any geometric form or form, including straight, branched, forked, etc., and "polyethylene glycol" as used herein is meant to encompass any water-soluble POLY (ethylene oxide). Typically, the PEG used in the present invention will comprise one of two of the following structures: "(CH)₂CH₂O)_k- "or" (CH)₂CH₂O)_k-CH₂CH₂- "depending on whether one or more of the terminal oxygens has been replaced, for example, during a synthetic transformation. The variable k ranges from 5 to about 500, and the terminal groups as well as the structure of the overall PEG can vary. The polyethylene glycol structure will typically also contain a partial terminal moiety residue, similar to the terminal group of POLY, which may be H, NH₂、OH、CO₂H、C_1-6Alkyl (e.g. methyl, ethyl, propyl), C_1-6Alkoxy (e.g., methoxy, ethoxy), acyl, or aryl.

Preferred "POLY" of the present invention are linear polyethylene glycols, typically of the structure:

represents a link of an atomJoint, mark "&The oxygen atom of the number "is the atom attached to the organic center" R ". Wherein k is in the range of about 5 to 500, preferably 50 to 200, r is any integer between 1 and 10, and more preferably, the term "POLY" according to the present invention is:

POLY of the present invention may also be:

and the like.

The active agent D is a camptothecin anticancer agent, and camptothecin drugs are clinical topoisomerase I inhibitors, have the defects of high activity, poor water solubility, high toxic and side effects on normal body tissues and the like, and greatly limit the clinical application of the camptothecin anticancer agent.

R in the structure of D₇Is a group covalently attached to a multivalent linker L, such as a hydroxyl, amino, or thiol, preferably a hydroxyl group. There should be no significant loss of biological activity when active agent D is attached to the multivalent linker L.

The active agent of the invention is preferably irinotecan, SN-38, 10-hydroxycamptothecin and rubitecan.

Wherein the structure of irinotecan is as follows:

the structure of SN-38 is as follows:

the structure of 10-hydroxycamptothecin is as follows:

rubitecan has the following structure:

in the invention, the T is a targeting molecule with or without medicinal effect, and the targeting molecule has the effect of increasing targeting property, so that the concentration of the conjugate in a target tissue is higher, and the physiological activity or the medicinal effect is improved. The "T" may be a monofunctional targeting molecule or a multifunctional targeting molecule, and in some alternatives, may also be a targeting moiety consisting of two or more targeting molecules. For example, T may be GE 11.

The polypeptide sequence of GE11 is: YHWYGYTPQNVI, its structure is:

the active agent "D" referred to herein refers to a portion of the unmodified parent active agent or the residue of the unmodified parent active agent prior to the covalent chain (or activated or chemically modified form thereof) resulting from covalent attachment of the drug to the multivalent linker of the present invention. The active agent itself is released upon hydrolysis or enzymatic hydrolysis of the linker between the active agent moiety and the multivalent linker.

For the purposes of the present invention, the term "residue" is understood to mean a part of a compound which is the residue after having undergone a substitution reaction with another compound.

When the conjugate of the present invention enters into an organism and reaches a target cell or a target tissue, the active agent D is cleaved from the polyvalent linker L, and the active agent D is released in a form in which it is not modified, i.e., a covalent bond is not formed, is separated from the parent body, and exerts physiological activity.

In a preferred embodiment of the invention, "POLY" is a linear polyethylene glycol linker arm, i.e., the conjugates of the invention include several types of compounds:

four arms:

three arms:

eight arms:

wherein k is in the range of about 5 to 500, preferably 50 to 200, and r is any integer between 1 and 10.

The compounds of the formula (III) are preferred according to the invention, and k is preferably 113 on the basis of the formula (III). It will be understood by those skilled in the art that in the polymer art, k represents the degree of polymerization of the polymer and is not an absolute number depending on the molecular weight of the polymer, and when k is 113, it means an average value of 113.

In a more preferred embodiment, the targeting moiety "T" of the conjugate of the invention is GE11 and the active agent "D" is selected from one of irinotecan, SN-38, 10-hydroxycamptothecin, rubitecan.

In a more preferred embodiment, L is selected from:

one kind of (1).

Based on formula (iii), in some particular embodiments, the compounds of the invention are as follows:

a compound f: d is irinotecan, T is GE11

Compound F is a pharmaceutically acceptable hydrochloride salt of compound F

More specifically, compound f can also be written as follows:

it is to be noted here that, in the salt formation, the side chain of the conjugate of the invention and HCl are respectively salified, for example, compound F, each side chain carries 3 molecules of HCl, and the whole molecule will carry 12 HCl molecules.

In addition to the specific compounds disclosed above, those skilled in the art can prepare more conjugates according to the technical scheme and preparation method of the present invention, such as:

d is SN-38, T is GE11 conjugate;

② D is 10-hydroxycamptothecin, T is GE11 conjugate;

③ D is rubitecan and T is GE11 conjugate.

The conjugate of the invention is a typical prodrug, and the active agent D is released through hydrolysis or enzymolysis, separated from a parent body and exerts physiological activity.

The conjugates of the invention exhibit high loading capacity, which allows for a reduction in the total dose to treat a particular disease, such as cancer, etc. That is, the active agent carriers of the conjugates of the invention are capable of effectively covalently bonding to a wide variety of active agent molecules, allowing a greater number of therapeutic dosage forms (i.e., active agent moieties) to be administered per a given amount of conjugate. The conjugate is modified by the water-soluble polymer, is hydrophilic in nature, and particularly improves the bioavailability of the conjugate when the active agent is a water-insoluble drug.

Compared with unconjugated drugs, the conjugate of the invention can show stronger effect and is more enriched in tissues in human bodies or other animal bodies.

The conjugate prodrugs of the present invention contain a number of unique properties, particularly where the active agent is an anti-cancer compound. The drug precursor can inhibit the growth of tumor with high efficiency. This small molecule we use is one known to have anti-cancer properties. However, by combining with multi-branched polymers as described above, the therapeutic efficacy and pharmacokinetics are greatly improved compared to the small molecule (e.g., the anti-cancer compound itself). Suitable solid tumor types include colon cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and malignant sarcomas, carcinomas, and lymphomas of the breast, ovary, colon, kidney, bile duct, lung, and brain.

In conclusion, the invention is a multi-arm polymer modified targeting anticancer conjugate, wherein the water-solubility polymer modification can enhance the water solubility of the conjugate, thereby improving the drug loading rate; the targeting molecule increases targeting property, so that the concentration of the conjugate in target tissues is higher; l is any connecting joint and has the function of connecting the targeting molecule and the anticancer drug firstly and then connecting the targeting molecule, the anticancer drug and the polymer arm, so that the whole conjugate forms an organic whole.

The conjugate of the invention, the pharmaceutically acceptable salt is preferably hydrochloride, can be salified by conventional means in the field of pharmaceutical chemistry, and can also be trifluoroacetate, sulfate, phosphate, acetate and the like.

In another aspect, the invention provides a method for preparing the conjugate. During the preparation of the conjugates of the invention, POLY and organic center R actually comprise a multi-armed polymer, which in the preferred embodiment of the invention is a multi-armed polyethylene glycol, and can be obtained from commercially available starting materials, such as various types of four-, three-, eight-armed polyethylene glycol derivatives available from Kyork technology, Inc., Beijing. These multi-arm PEGs, which are commercially available, can participate directly in the reaction.

In preparing the conjugate of formula (III), the four-armed polyethylene glycol preferably used is as follows:

the preferred four-ARM polyethylene glycol is referred to as 4ARM-PEG20K-SCM and has a molecular weight of about 20 kDa. Similarly, in the preparation of the conjugates of formula (IV) and formula (V), the three-and eight-arm polyethylene glycols used also preferably have a molecular weight of about 20 kDa.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention will be described in detail below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are provided for the purpose of making the disclosure more complete and complete. The reagents and raw materials are commercially available except for the preparation method, wherein 4ARM-PEG20K-SCM is available from Kyork technology Co., Ltd

Noun interpretation

DMF: n, N-dimethylformamide

DCM: methylene dichloride

Boc-Gly-OH：

DMAP: 4-dimethylaminopyridine

DCC: dicyclohexylcarbodiimide

IPA: isopropanol (I-propanol)

TFA: trifluoroacetic acid

TBME: tert-butyl methyl ether

EA: ethyl acetate

DME: ethylene glycol dimethyl ether

Fmoc-OSU: 9-fluorenylmethyl-N-succinimidyl carbonate

THF: tetrahydrofuran (THF)

H-Lys(Boc)-OBzl·HCl：

DIEA: n, N-diisopropylethylamine

DEPC: cyanophosphoric acid diethyl ester

DEA: triethylamine

Pbf：

HOBT: 1-hydroxybenzotriazoles

DIC: n, N-diisopropylcarbodiimide

TFE: trifluoroethanol

DPPA: azoic acid diphenyl ester

SPPS: solid phase organic synthesis

NMM: n-methylmorpholine

And (3) TIS: tri-isopropyl silane

MTBE: tert-butyl methyl ether

Example 1

Preparation of Compound 2

Adding 3.50g of compound 1(1.0eq) and 52.5mL of DMF into a 250mL round-bottom flask, heating to 60 ℃ for dissolution, distilling off the DMF under reduced pressure after 5-10min, adding 300mL of n-heptane, distilling under reduced pressure for three times, adding 105mL of DCM, 1.08g of Boc-Gly-OH (1.2eq) and 63mg of DMAP (0.1eq) after spin-drying, adding 1.59g of DCC (1.5eq) dissolved in 10mL of DCM, reacting at 20 ℃ for 4 hours, filtering after TLC monitoring reaction, adding 120mL of IPA when the reaction is finished, distilling off 75% of the solvent, adding 150mL of n-heptane, stirring at room temperature for 1 hour, filtering, washing with n-heptane for 2 times, and drying to obtain 4.02g of compound 2 as a light yellow solid.

Preparation of Compound 3

Adding 4.02g of compound 2 and 50mL of DCM into a 100mL three-necked flask, stirring and dissolving, then adding 11.6mL of TFA dropwise, reacting for 2h at room temperature, monitoring by TLC, adding 150mL of acetonitrile after the reaction is finished, distilling 120mL of solvent under reduced pressure, pouring into 320mL of TBME solution, stirring for 30min, filtering, and washing a filter cake with TBME to obtain 4.00g of light yellow solid compound 3.

Example 2

Preparation of Compound 5

Adding 6.9g of compound 4 and 30mL of EA into a 250mL three-necked flask, stirring to dissolve, cooling to 0 ℃, adding 40mL of 0.3M HCl/EA, keeping the temperature for reaction for 2 hours, and concentrating to dryness after the TLC monitoring reaction to obtain a compound 5, and directly carrying out the next reaction.

Preparation of Compound 6

Dissolving compound 5(1.0eq) with 50ml of purified water, adding 3.96g of sodium bicarbonate (2.0eq), dissolving 5.30g of Fmoc-OSU (1.0eq) with 50ml of DME, adding into a reaction flask of compound 5, supplementing with 25ml of THF, stirring at room temperature for 2 hours, after TLC monitoring reaction is finished, evaporating organic solvent, EA extracting impurities, adjusting pH of an aqueous phase to 3-4 with dilute hydrochloric acid, EA extracting for 2 times, combining organic phases, washing with water once, washing with saturated saline, drying with anhydrous sodium sulfate, and concentrating to obtain 8.4g of light yellow oily compound 6.

Preparation of Compound 7

In a 100ml reaction flask were charged 4.00g of Compound 6(1.0eq), 2.92g H-Lys (Boc) -OBzl & HCl, and 40ml of DCM to dissolve them, and 2.76g of DIEA (3.0eq), 1.74g of DEPC (1.5eq) were added thereto, and the mixture was stirred at room temperature for 2 hours, after the TLC-monitored reaction was completed, it was washed with an aqueous acetic acid solution, with a sodium hydrogencarbonate solution, once with water, once with a saturated saline solution, and then dried over anhydrous sodium sulfate, and concentrated to obtain 7.0g of Compound 7 as a pale yellow oil, which was then subjected to the next reaction without being purified. (Compound 16 was prepared by the same method)

Preparation of Compound 8

After completion of TLC monitoring reaction, the mixture was concentrated to dryness, 100ml of 50ml of EA was added thereto, pH was adjusted to 3-4 with dilute hydrochloric acid, liquid separation was performed, and the aqueous phase was extracted with EA for 2 times and concentrated to dryness to obtain 3.5g of Compound 8 as a pale yellow solid. (Compound 17 was prepared by the same method)

Example 3

Preparation of protecting group-attached targeting molecule GE11 (Compound 60)

Sequence YHWYGYTPQNVI of GE11

The following protected amino acids were attached to the Resin in sequence using 2Cl-Trt Resin, HOBT/DIC as coupling reagent, DMF as reaction solvent, ninhydrin assay for reaction monitoring: Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Pro-OH, Fmoc-Thr (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH, Boc-Tyr (tBu) -OH, addition of a cleavage reagent: acetic acid/TFE/DCM ═ 1/2/7, reaction for 2 hours, precipitation with ice MTBE, washing, and drying to give off-white solid 60.

EXAMPLE 4 preparation of Compound F and Compound F

Preparation of Compound 61

5.00g of compound 60(1.0eq), 1.66g of compound 17(1.1eq), 50mL of DMF, 0.72g of DIEA (3.0eq), 0.45g of DEPC (1.5eq) are added into a 100mL reaction flask, and reacted at room temperature for 2 hours, after the TLC reaction is finished, the mixture is poured into 10mL of water, EA is extracted for 2 times, acetic acid aqueous solution is washed, sodium bicarbonate solution is washed, saturated sodium chloride is washed, anhydrous sodium sulfate is dried, and jelly-like solid 616.14 g is obtained by concentration, and the next reaction is directly carried out.

Preparation of Compound 62

Adding 6.10g of compound 61, 150ml of methanol and 0.31g of Pd/C into a 200ml hydrogenation kettle, hydrogenating overnight, filtering after TLC reaction is finished, and concentrating to obtain 625.98 g of gray solid compound

Preparation of Compound 63

5.95g of compound 62(1.0eq), 1.36g of compound 3(1.05eq), 60ml of DMF, 0.68g of DIEA (3.0eq), 0.43g of DEPC (1.5eq) are added into a 100ml reaction flask, the mixture is reacted for 2 hours at room temperature, the mixture is poured into 600ml of TBME after TLC reaction is finished, the mixture is pumped and filtered after being pulped, the solid is dissolved by 100ml of DCM and poured into 1.0L of TBME, the mixture is pumped and filtered after being pulped, 636.53 g of gray powder compound is dried, and the next reaction is directly carried out.

Preparation of Compound 64

6.50g of Compound 63 130ml of lysis reagent: acetic acid/TFE/DCM ═ 1/2/7, reaction 2 hours, precipitation with ice MTBE, washing, drying and HPLC purification gave 3.63g of off-white solid compound 64. Preparation of Compound 65

2.06g of compound 64(4.5eq), 3.00g of 4ARM-PEG20K-SCM (1.0eq), 30ml of DMF (dimethyl formamide), 0.13g of TEA (9.0eq) are added into a 50ml reaction bottle to react at room temperature, after the reaction is monitored by HPLC to have no obvious progress, the mixture is poured into 300ml of TBME, pulped, filtered by suction and dried to obtain 654.67 g of compound, and the compound is directly used for the next reaction.

Synthesis of Compound f

Into a 200ml reaction flask, 4.60g of compound 65, 100ml lysis reagent: 92.5% TFA/2.5% water/2.5% TIS, stirred at room temperature for 2h, precipitated with ice MTBE, centrifuged, washed, crude product purified by reverse phase HPLC, desalted, concentrated to remove organic solvent, and lyophilized to give 1.34g of off-white powder compound f.

Synthesis of Compound F

And (3) purifying the crude compound F by reverse phase HPLC, desalting, concentrating to remove the organic solvent, adjusting the pH value to 5-6 by using dilute hydrochloric acid, and freeze-drying to obtain 1.34g of a yellow-green powder compound F.

MALDI-TOF molecular weight 30907.82.

EXAMPLE 5 evaluation of the in vivo efficacy of Compounds in HT-29 nude mouse graft tumor model

1. Purpose of experiment

Evaluation of efficacy of Compound F and Compound F following administration in mouse animal models of xenografted BALB/c nude of HT-29 cell line of human Colon cancer.

2. Experimental Material

2.1 test article

Irinotecan (bulk drug) was purchased and nktr-102 and 2 compounds tested were provided by borrelid biopharmaceutical (suzhou) corporation.

The preparation method of nktr-102 refers to the method disclosed in CN102711837A, and comprises the following steps:

compound 3(829mg,4.5eq) from example 1 was added to a 250mL reaction flask, DCM (50mL), triethylamine (221mg,9.0eq) were added, and after dissolution 4ARM-PEG20K-SCM (5.00g,1.0eq) was added to the reaction flask. After no significant progress of the reaction was monitored by HPLC, about 20mL of DCM was distilled off under reduced pressure, the solution was poured into 300mL of TBME and precipitated with stirring, filtered to give 5.4g of crude product, which was purified by HPLC preparative, desalted, adjusted to pH 5-6 with dilute hydrochloric acid, and lyophilized to give 2.71g of light green powder nktr-102.

2.2 reagents

McCoy's 5A culture solution, Fetal Bovine Serum (FBS), trypsin, cyan-chain double antibody, water for injection, normal saline, lactic acid and sorbitol.

2.3 Experimental animals

Female BALB/c nude mice (the number is 150; the week age is 6-7 weeks) are purchased from Beijing Wintolite laboratory animal technology Limited, and are bred in an SPF animal room, the temperature is 20-25 ℃, the relative humidity is 40-70%, and the illumination is carried out for 12 hours respectively; animals had free access to water and food. After about 1 week of normal feeding, mice with good signs can be selected for this experiment by veterinary examination. Marking the tail and root of the animals by using a marker pen before grouping, and marking each animal after grouping by using an ear clipping mode.

2.4 transplantable tumor strains

Human colon cancer cells HT-29, from the cell bank of the Committee for culture Collection of the Chinese academy of sciences (CAS, the laboratory liquid Nitrogen cryopreservation).

3. Experimental methods

3.1HT-29 cell culture

At 5% CO₂HT-29 cells are subjected to conventional cell culture in a culture solution containing 10% fetal bovine serum McCoy's 5A under the culture condition of 37 ℃; passage with 0.25% pancreatin; according to the growth condition of the cells, the cells are passaged 2 to 3 times per week at the passage ratio of 1:4 to 1: 6.

3.2 animal model preparation

Collecting HT-29 cells in logarithmic growth phase, counting the cells, suspending in serum-free McCoy's 5A culture medium, and adjusting the cell concentration to 4 × 10⁷cell/mL; blowing and beating the cells by using a pipettor to disperse the cells uniformly, then putting the cells into a 50mL centrifuge tube, and putting the centrifuge tube into an ice box; the cell suspension was aspirated by a 1mL syringe, injected subcutaneously into the right forelimb armpit of nude mice, and inoculated with 100. mu.L (4X 10) per animal⁶Cell/cell), establishing a HT-29 nude mouse transplantation tumor model. After inoculation, the animal state and the tumor growth condition are regularly observed, the tumor diameter is measured by using an electronic vernier caliper, the data is directly input into an Excel spreadsheet, and the tumor volume is calculated. When the tumor volume reaches 100-300 mm³30 animals with good health status and similar tumor volume were selected and divided into 5 groups (n is 6) by the randomized block method. Tumor size was measured 2 times per week after the start of the experiment, tumor volume was calculated, and animal body weight was weighed and recorded.

Tumor Volume (TV) calculation formula is as follows:

TV(mm³)＝l×w²/2

wherein l represents the tumor major axis (mm); w represents the tumor minor diameter (mm).

3.3 preparation of solvent

0.5g of sorbitol was weighed into a 50mL centrifuge tube, 50mL of water for injection was added to the centrifuge tube, and the solid matter was dissolved completely by vortex oscillation to prepare a 1% sorbitol aqueous solution (w/v) and stored in a refrigerator at 4 ℃ for future use.

3.4 preparation of drug delivery preparation

3, 4.1 irinotecan administration preparation

12.0mg of irinotecan was weighed, 0.15mL of 1% lactic acid was added, the mixture was vortexed to completely dissolve the drug, 2.85mL of 1% sorbitol aqueous solution was added, and the mixture was vortexed and mixed uniformly, whereby the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The effective concentration of irinotecan in the solution is 4.0 mg/mL^-1。

3.4.2nktr-102 administration preparation

Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.5mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, whereby the effective concentration of irinotecan in the solution was 4.0 mg. multidot.mL^-1。

3.4.3 formulation of administration of Compound a and Compound A

Before each administration, respectively accurately weighing 120.3mg of compound a and compound A, adding 2.5mL of physiological saline, vortex vibrating, and ultrasonically (if necessary) completely dissolving the medicine, wherein the effective concentration of irinotecan in the solution is 4.0 mg/mL^-1。

3.4.4 formulation of Compound F and Compound F for administration

Before each administration, 145.9mg of compound F is accurately weighed, 2.5mL of physiological saline is added, vortex oscillation and ultrasonic treatment (if necessary) are carried out to completely dissolve the medicine, and the effective concentration of irinotecan in the solution is 4.0 mg/mL^-1。

3.5 animal grouping and administration

Animal groups and dosing regimens are shown in table 1. The first administration is started on the grouping day, the experiment is finished after about 21 days, and the administration volumes are 10 mL/kg^-1. The effective dose of the reduced irinotecan is 40mg kg^-1. The first group was a solvent control group, and the saline was administered to the tail vein 1 time every 4 days for 3 times (Q4D × 3). In groups 2 to 5, test samples of irinotecan, nktr-102, compound F, compound F were administered by tail vein injection, each once every 4 days, Q4D X3.

TABLE 1 drug effect experiment dosing regimen for nude mouse transplanted tumor model

3.6 end of experiment

On the last day of the experiment, the animals were weighed, and euthanized after tumor size measurement (CO)₂). And stripping and weighing tumor tissues, and calculating the tumor weight inhibition rate.

4. Data recording, calculation formula

The formula for the Relative Tumor Volume (RTV) is:

RTV＝TV_t/TV_initial

wherein, TV_initialTumor volume measured when administered in groups; TV (television)_tThe tumor volume at each measurement during dosing.

The relative tumor proliferation rate (% T/C) was calculated by the following formula:

％T/C＝100％×(RTV_T/RTV_C)

wherein, RTV_TRepresenting treatment group RTV; RTV_CRepresenting the solvent control RTV.

The calculation formula of the tumor growth inhibition rate TGI (%) is as follows:

TGI＝100％×[1－(TV_t(T)－TV_initial(T))/(TV_t(C)－TV_initial(C))]

wherein, TV_t(T)Represents the tumor volume for each measurement in the treatment group; TV (television)_initial(T)Represents the tumor volume of the treatment group when administered in groups; TV (television)_t(C)Represents the tumor volume of each measurement of the solvent control group; TV (television)_initial(C)The tumor volume of the solvent control group at the time of group administration is indicated.

The formula for calculating the weight loss rate of the animals is as follows:

weight loss rate of animal (BW) 100 × (BW)_initial－BW_t)/BW_initial

Wherein, BW_tRepresents the animal body weight measured at each time during the dosing period; BW (Bandwidth)_initialThe body weight of the animals at the time of the group administration is indicated.

The calculation formula of the tumor weight inhibition rate IR (%) is as follows:

IR(％)＝100％×(W_C－W_T)/W_C

wherein, W_CRepresenting tumor weight of control group; w_TIndicates the tumor weight of the treated group.

5. Statistical analysis method

Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. Data are expressed as Mean ± standard error (Mean ± SE) and comparisons between groups are performed using the t-test, unless otherwise specified.

6. Experimental observation

During the course of the experiment, the laboratory and veterinarians need to continuously observe the signs and health of the experimental animals. Any abnormal manifestations of the animal, such as pain, depression, decreased activity, etc., need to be recorded in the original record of the experiment. If the abnormal performance of the experimental animal exceeds the welfare of the IACUC-related animal, the veterinarian can determine whether to terminate the experiment and notify the responsible person of the experimental project.

7. Results

For the human cancer xenograft tumor model, the relative tumor proliferation rate T/C (%) is recommended as the test evaluation index, and the lower the proliferation rate, the better the tumor inhibition effect, see table 2.

TABLE 2 tumor proliferation rate T/C (%)

P <0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102

# P <0.05 compared to% T/C for the blank solvent, irinotecan, and nktr-102 group

Experimental results show that the compound has good inhibition effect on the in-vivo growth of human colon cancer HT-29 nude mouse transplantation tumor model tumor, and is superior to irinotecan and nktr-102.

Example 6 inhibition of human Breast cancer MDA-MB-231 nude mouse xenograft model

1. Purpose of experiment

This study evaluated the in vivo anti-tumor activity of compound F and compound F using a human breast cancer MDA-MB-231 nude mouse graft tumor model.

2. Laboratory animal

2.1 animal species

A mouse.

2.2 variety

BALB/c nude mice.

2.3 sex

And (4) female.

2.4 number

150 were inoculated and 30 were used for the experiment.

Age 2.5

6-8 weeks.

2.6 body weight

20-22 g. + -. 20% body weight mean.

2.7 animal sources (suppliers)

Shanghai Sphere-BiKai laboratory animals Co., Ltd (BK), license number SCXK (Shanghai) 2008-0016.

2.8 management of laboratory animals

All experimental animals were housed in an SPF scale laboratory. The experimenters were responsible for routine care and experimental studies.

2.8.1 animal identity identification method

Each mouse cage is hung with an identity card with information such as experiment number, experiment group, name of experimenter, mouse variety and sex, and the mouse is marked with an ear nail.

2.8.2 random grouping

When the tumor volume reaches 150-³Then dividing the mice into 5 groups by a random block method, wherein each group comprises 6 mice, and ensuring that the tumor volume and the weight of the mice among the groups are uniform. The mean tumor volumes of the groups differed from the mean tumor volumes of all experimental animals by no more than ± 10%.

2.8.3 operation management Specification

All experimental animals strictly adhere to the guidelines for animal use and management.

2.8.4 feeding conditions

The living conditions are as follows: IVC system, 6 per cage

Temperature: 20-26 deg.C

Humidity: 40% +/-70%

Illumination: 12 hours of day and night alternation

2.8.5 feed

Irradiated rat and rat feeds were purchased from Aojieli feeds, Inc., Beijing, Ke. Free food intake.

2.8.6 Drinking water

City tap water is filtered, autoclaved and sterilized for drinking.

2.8.7 bedding

Corncobs, Shanghai Miao derivatives science and technology Co., Ltd, were used after autoclaving. The pads were changed twice a week.

2.8.8 acclimation period

Mice were given a minimum one week environmental acclimation period prior to the experiment.

3. Experimental Material

3.1 test drugs

3.2 other chemical reagents and materials

3.2.1 physiological saline

Physiological saline was purchased from Shanghai Huayuan Changfu pharmaceutical industry (group) Co., Ltd.

3.2.2 sterile Syringe

A1 ml sterile syringe was purchased from Shanghai Kangdelai Enterprise development group, Inc. (Shanghai, China).

3.2.3 cell lines

Human breast cancer MDA-MB-231 was purchased from the Shanghai institute of cell biology.

MDA-MB-231 was cultured in DMEM medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and in 5% CO₂37 ℃ incubator.

3.2.4 Matrigel (BD Matrigel)

Matrigel was purchased from BD corporation, usa

3.3 instruments

Biosafety cabinets (model: AC2-6E1), available from ESCO;

CO₂a water-tight cell culture chamber (model: 3111) from Thermo Scientific Forma;

an inverted microscope (model: CKX41SF) from Olympus;

electric suction apparatus (model YX930D), available from Shanghai medical devices industry (group) Inc.;

a balance (Mettler-Torloduo AB135-S) available from Mettler-Torluo;

low speed centrifuge (model LD5-2A) available from Beijing Rebo centrifuge, Inc.;

an electronic digital display caliper (model: SF2000) purchased from Guilin Guangdong digital measurement and control GmbH.

4. Design of experiments

Establishing a model of human breast cancer MDA-MB-231 nude mice subcutaneous transplantation tumor, wherein each nude mouse is inoculated with 1 × 10⁶And (4) cells.

The following (table 3) doses and dosing schedules were designed for this trial.

Table 3: anti-tumor effect of human breast cancer MDA-MB-231 in nude mouse transplantation tumor model

5. Compound preparation

The formulation method is provided by borry biomedical technology (Suzhou) Inc.

3mL volume required for a single administration.

5.1 formulation of irinotecan administration preparation

12.0mg of irinotecan was weighed, 0.15mL of 1% lactic acid was added, the mixture was vortexed to completely dissolve the drug, 2.85mL of 1% sorbitol aqueous solution was added, and the mixture was vortexed and mixed uniformly, whereby the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The concentration of irinotecan in the free form in the solution was 4.0 mg/mL^-1。

5.2nktr-102 dosing formulations

Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.5mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, and the concentration of irinotecan in the solution was 4.0 mg. multidot.mL in the free form^-1。

5.3 preparation of compound F and compound F for administration: accurately weighing, adding 2.5mL of physiological saline, and vortex vibrating to completely dissolve the medicine, wherein the free form concentration of irinotecan in the solution is 4.0 mg/mL^-1。

6. Experimental methods

MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA). Cells were placed in 5% CO₂The culture was carried out in an incubator at 37 ℃.

Establishing a tumor nude mouse subcutaneous transplantation model by a cell inoculation method: collecting tumor cells in logarithmic growth phase, counting, re-suspending in 1 × PBS, and adjusting cell suspension concentration to 1 × 10⁷And/ml. The right dorsal part of the nude mice was inoculated subcutaneously with 1X 10 tumor cells using a 1ml syringe (No. 4 needle)⁶0.1 ml/mouse.

The tumor volume reaches 100-200mm³When the animals are divided into 5 groups according to a random block method, the tumor difference of each group is less than 10 percent of the mean value, 6 animals in each group are divided into 6 animals, the current Day is used as Day1, and the animals are administrated on the current Day.

The experimental period was carried out for 3 weeks, during which animal body weight and tumor size were measured twice a week. Clinical symptoms were recorded with daily observations. Animals were sacrificed the last day of the experiment, weighed, tumors stripped, weighed and photographed for documentation.

All animal experimental procedures strictly adhere to animal use and management regulations. The calculation of the tumor related parameters refers to the CFDA technical guidance principle of non-clinical research of cytotoxic antitumor drugs in China.

The Tumor Volume (TV) is calculated as: TV as a × b²/2. Where a, b represent tumor measurement length and width, respectively. The Relative Tumor Volume (RTV) is calculated as: RTV-Vt/V₀. Wherein V₀Tumor volume at the time of group administration and Vt is the tumor volume at the time of measurement. Having antitumor activityThe evaluation indexes are relative tumor increment rate T/C (%) and tumor inhibition rate (%), and the calculation formulas are respectively as follows: T/C (%) ═ T_RTV/C_RTV)×100％。T_RTVTo the treatment group RTV, C_RTVNegative control group RTV; tumor inhibition (%) (average tumor weight in negative control group-average tumor weight in administration group)/average tumor weight in negative control group × 100%.

The change (%) in body weight of tumor-bearing animals was calculated as follows: (weight-at-measurement-weight-at-group)/weight-at-group × 100.

7. Data analysis

8. Results and reports

According to the Chinese CFDA non-clinical research technical guide principle of cytotoxic antitumor drugs (11 months 2006), T/C (percent) is less than or equal to 40 percent, and p is less than 0.05 through statistical analysis to be effective, which is shown in Table 4.

TABLE 4 tumor proliferation rate T/C (%)

P <0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102

Experimental results show that the compound has good inhibition effect on human breast cancer MDA-MB-231 nude mouse transplanted tumor, and is superior to irinotecan and nktr-102.

Example 7 inhibition of human pancreatic cancer MIA Paca-2 nude mouse xenograft model

1. Purpose of experiment

This study evaluated the in vivo anti-tumor activity of compound F and compound F using a human pancreatic cancer MIA Paca-2 nude mouse transplantation tumor model.

2. Laboratory animal

2.1 animal species

A mouse.

2.2 variety

BALB/c-nu/nu nude mice.

2.3 sex

And (4) female.

2.4 number

150。

2.5. Age (age)

6-8 weeks.

2.6. Body weight

20-22 g. + -. 20% body weight mean.

2.7. Animal origin (supplier)

2.8. Laboratory animal management

All experimental animals were housed in an SPF scale laboratory.

2.8.1 animal identity identification method

2.8.2 random grouping

2.8.3 operation management Specification

The operation and management of all experimental animals strictly comply with the guiding principles of the use and management of the experimental animals.

2.8.4 feeding conditions

The living conditions are as follows: IVC system, 6 per cage

Temperature: 25 ℃ plus or minus 1 DEG C

Humidity: 65% +/-10%

Illumination: 12 hours of day and night alternation

2.8.5 feed

2.8.6 Drinking water

City tap water is filtered, autoclaved and sterilized for drinking.

2.8.7 bedding

2.8.8 acclimation period

3. Experimental Material

3.1 test drugs

3.2 other chemical reagents and materials

3.2.1 physiological saline

Physiological saline was purchased from Shanghai Huayuan Changfu pharmaceutical industries (group) Co., Ltd. (Shanghai, China).

3.2.2 sterile Syringe

3.2.3 cell lines

Human pancreatic cancer MIA Paca-2 was purchased from Shanghai institute of sciences cell biology.

MIA Paca-2 was cultured in DMEM medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and 2.5% HS in 5% CO₂37 ℃ incubator.

3.2.4 Matrigel (BD Matrigel)

Matrigel was purchased from BD corporation, usa

3.3 instruments

Biosafety cabinets (model: AC2-6E1), available from ESCO;

an inverted microscope (model: CKX41SF) from Olympus;

a balance (Mettler-Torloduo AB135-S) available from Mettler-Torluo;

4. Design of experiments

Establishing a human pancreatic cancer MIA Paca-2 nude mouse subcutaneous transplantation tumor model, and inoculating 3X 10 tumor cells to each nude mouse⁶And (4) cells.

The following (table 5) doses and dosing schedules were designed for this trial.

Table 5: antitumor effect in human pancreatic cancer MIA Paca-2 nude mouse transplantation tumor model

Tumor strain: MIA Paca-2; a total of 30 inoculations

5. Compound preparation

The formulation method is provided by borry biomedical technology (Suzhou) Inc.

A single administration requires a volume of 3 mL.

5.1 formulation of irinotecan administration preparation

5.2nktr-102 dosing formulations

Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.5mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, and the concentration of irinotecan in the solution was 4.0 mg. multidot.mL-1 in the free form.

5.3 Compounds f andf, preparation of a dosing preparation: accurately weighing, adding 2.5mL of physiological saline, and vortex vibrating to completely dissolve the medicine, wherein the free form concentration of irinotecan in the solution is 4.0 mg/mL^-1.6. Experimental methods

MIA Paca-2 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA) and 2.5% HS. Cells were placed in 5% CO₂The culture was carried out in an incubator at 37 ℃.

Establishing a tumor nude mouse subcutaneous transplantation model by a cell inoculation method: collecting tumor cells in logarithmic growth phase, counting, re-suspending in 1 × PBS, and adjusting cell suspension concentration to 3 × 10⁷And/ml. Nude mice were inoculated subcutaneously on the right dorsal side with tumor cells, 3X 10, using a 1ml syringe (No. 4 needle)⁶0.1 ml/mouse.

All animal experimental procedures strictly adhere to animal use and management regulations. The calculation of the tumor related parameters refers to the SFDA non-clinical research technical guidance principle of cytotoxic antitumor drugs in China.

The Tumor Volume (TV) is calculated as: TV as a × b²/2. Where a, b represent tumor measurement length and width, respectively. The Relative Tumor Volume (RTV) is calculated as: RTV-Vt/V₀. Wherein V₀Tumor volume at the time of group administration and Vt is the tumor volume at the time of measurement. The evaluation indexes of the antitumor activity are relative tumor increment rate T/C (%) and tumor inhibition rate (%), and the calculation formulas are respectively as follows: T/C (%) ═ T_RTV/C_RTV)×100％。T_RTVTo the treatment group RTV, C_RTVNegative control group RTV; tumor inhibition ratio (%) (average tumor weight in negative control group-average tumor weight in administration group)/average tumor weight in negative control group100％。

According to the Chinese SFDA non-clinical research technical guide principle of cytotoxic antitumor drugs (11 months 2006), T/C (percent) is less than or equal to 40 percent, and P <0.05 is analyzed by statistics to be effective.

7. Data analysis

Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. Data are expressed as Mean ± standard error (Mean ± SE) unless otherwise stated, and comparisons between groups were by t-test with P <0.05 as a significant difference.

8. Results and reports

According to the Chinese CFDA non-clinical research technical guide principle of cytotoxic antitumor drugs (11 months 2006), T/C (percent) is less than or equal to 40 percent, and P <0.05 is analyzed to be effective by statistics, which is shown in Table 6.

TABLE 6 tumor proliferation rate T/C (%)

P <0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102

Experimental results show that the compound has good inhibition effect on human pancreatic cancer MIA Paca-2 nude mouse transplantation tumor, and is superior to irinotecan and nktr-102.

Example 8 inhibition of tumor growth in a nude mouse transplanted tumor model of human gastric cancer NCI-N87 cell line.

1. Purpose of experiment

The inhibitory effect of the test drugs compound F and compound F on the in vivo growth of the tumor of the nude mouse transplanted tumor model of the human gastric cancer NCI-N87 cell line is evaluated.

2. Experimental Material

2.1 test article

Irinotecan (bulk drug) was purchased and, thus, nktr-102 and 2 compounds tested were provided by borrelid biopharmaceutical (suzhou) corporation.

2.2 reagents

RPMI-1640 culture medium, Fetal Bovine Serum (FBS), trypsin, cyan-chain double antibody and normal saline.

2.3 Experimental animals

Female BALB/c nude mice (the number is 150; the week age is 6-8 weeks) are purchased from Beijing Wintolite laboratory animal technology, Inc., and are bred in an SPF animal house of St.Su.Sco, Suzhou, the temperature is 20-25 ℃, the relative humidity is 40-70%, and the illumination is carried out for 12 hours respectively; animals had free access to water and food. After about 1 week of normal feeding, mice with good signs can be selected for this experiment by veterinary examination. Marking the tail and root of the animals by using a marker pen before grouping, and marking each animal after grouping by using an ear clipping mode.

2.4 transplantable tumor strains

Human gastric cancer cell NCI-N87, derived from the cell bank of the Committee for culture Collection of the Chinese academy of sciences (CAS, the laboratory is frozen in liquid nitrogen).

3 Experimental methods

3.1 NCI-N87 cell culture

At 5% CO₂Under the culture condition of 37 ℃, NCI-N87 cells are cultured in a culture solution containing 10 percent fetal calf serum RPMI-1640 by a conventional method; passage with 0.25% pancreatin; according to the growth condition of the cells, the cells are passaged 1 to 2 times per week at the passage ratio of 1:2 to 1: 6.

3.2 animal model preparation

Collecting NCI-N87 cells in logarithmic growth phase, counting the cells, suspending in serum-free RPMI-1640 medium, and adjusting the cell concentration to 5 × 10⁷cell/mL; blowing and beating the cells by using a pipettor to disperse the cells uniformly, then putting the cells into a 50mL centrifuge tube, and putting the centrifuge tube into an ice box; the cell suspension was aspirated by a 1mL syringe and injected subcutaneously into the right forelimb axilla of nude mice, one animalInoculation with 100. mu.L (5X 10)⁶Cell/cell), establish NCI-N87 nude mouse graft tumor model. After inoculation, the animal state and the tumor growth condition are regularly observed, the tumor diameter is measured by using an electronic vernier caliper, the data is directly input into an Excel spreadsheet, and the tumor volume is calculated. When the tumor volume reaches 100-300 mm³30 animals with good health status and similar tumor volume were selected and divided into 5 groups (n is 6) by the randomized block method. Tumor size was measured 2 times per week after the start of the experiment, tumor volume was calculated, and animal body weight was weighed and recorded.

Tumor Volume (TV) calculation formula is as follows:

TV(mm³)＝l×w²/2

3.3 preparation of solvent

3.4 preparation of drug delivery preparation

3, 4.1 irinotecan administration preparation

3.4.2nktr-102 administration preparation

Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.3mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, and the concentration of irinotecan in the solution was 4.0 mg. multidot.mL in the free form^-1。

3.4.3 preparation of compound F, compound F for administration: accurately weighing, adding 2.5mL of physiological saline, and vortex vibrating to completely dissolve the irinotecan in the solution at a free form concentration of 4.0 mg/mL^-1。

3.5 animal grouping and administration

Animal groups and dosing regimens are shown in table 7. The first administration was started on the day of grouping, and the experiment was ended after 21 days, and the administration volumes were all 10 mL/kg^-1. Group 1 was a solvent control group, and the solvent was administered to the tail vein by injection 1 time every 4 days for 3 times (Q4D × 3). In groups 2 to 5, test samples of irinotecan, nktr-102 and the test compound were administered by tail vein injection at a dose of 40 mg/kg^-1(calculated as irinotecan content), Q4D × 3.

TABLE 7 drug effect experiment dosing regimen for nude mouse transplanted tumor model

3.6 end of experiment

After the experiment was completed, the animals were weighed, and the animals were euthanized after tumor size measurement (CO)₂). And stripping and weighing tumor tissues, and calculating the tumor weight inhibition rate. Tumor tissues were weighed and transferred to a sub-70 ℃ freezer for storage for subsequent analysis.

4. Data recording, calculation formula

The formula for the Relative Tumor Volume (RTV) is:

RTV＝TV_t/TV_initial

％T/C＝100％×(RTV_T/RTV_C)

TGI＝100％×[1－(TV_t(T)－TV_initial(T))/(TV_t(C)－TV_initial(C))]

The formula for calculating the weight loss rate of the animals is as follows:

weight loss rate of animal (BW) 100 × (BW)_initial－BW_t)/BW_initial

IR(％)＝100％×(W_C－W_T)/W_C

5. Statistical analysis method

Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. Data are expressed as Mean ± standard error (Mean ± SE) and the two comparisons are performed using t-test, unless otherwise specified.

6. Experimental observation

7. Results

For the human cancer xenograft tumor model, the relative tumor proliferation rate T/C (%) is recommended as the test evaluation index, and the lower the proliferation rate, the better the tumor inhibition effect, see table 8.

TABLE 8 tumor proliferation rate T/C (%)

P <0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102

Experimental results show that the compound has good inhibition effect on tumor growth of a nude mouse transplantation tumor model of human gastric cancer NCI-N87 cell strain, and is superior to irinotecan and nktr-102.

Example 9 effect on survival of U87MG nude mouse brain in situ model.

1. Purpose of experiment

The effect of test agents compound F and compound F on the survival rate of U87MG nude mouse brain in situ model was evaluated.

2. Experimental Material

2.1 test article

2.2 reagents

RPMI-1640 culture medium, trypsin, cyan-catenin and normal saline.

2.3 Experimental animals

Female BALB/c nude mice (the number is 150; the week age is 6-8 weeks) are purchased from Beijing Wintolite laboratory animal technology Limited, and are bred in an SPF animal room, the temperature is 20-25 ℃, the relative humidity is 40-70%, and the illumination is carried out for 12 hours respectively; animals had free access to water and food. After about 1 week of normal feeding, mice with good signs can be selected for this experiment by veterinary examination. Marking the tail and root of the animals by using a marker pen before grouping, and marking each animal after grouping by using an ear clipping mode.

2.4 transplantable tumor strains

Glioma cells U87MG, derived from the cell bank of the culture Collection of the type (CAS, the laboratory liquid nitrogen frozen stock) of Chinese academy of sciences.

3. Experimental methods

NCI-N87 cell culture

At 5% CO₂Under the culture condition of 37 ℃, NCI-N87 cells are cultured in RPMI-1640 culture solution in a conventional way; passage with 0.25% pancreatin; according to the growth condition of the cells, the cells are passaged 1 to 2 times per week at the passage ratio of 1:2 to 1: 6.

3.1 animal model preparation

Collecting NCI-N87 cells in logarithmic growth phase, counting the cells, suspending in serum-free RPMI-1640 medium, and adjusting the cell concentration to 1 × 10⁸cell/mL; blowing and beating the cells by using a pipettor to disperse the cells uniformly, then putting the cells into a 50mL centrifuge tube, and putting the centrifuge tube into an ice box; the cell suspension is sucked by a 1mL syringe, and 1 mu L (1X 10) of the human brain glioma cell U87MG cells cultured in vitro is guided by an animal stereotaxic apparatus by a microinjection method⁵Cell/cell), a U87MG brain glioma in situ model was established, and the animal status was observed periodically after inoculation. On day 12 post inoculation, 30 animals were selected and divided into 5 groups (n-6) using the randomized block method.

3.2 preparation of drug delivery preparation

3.2.1 formulation of irinotecan administration preparation

3.2.2nktr-102 dosage formulations

3.2.3 preparation of administration of Compound F, Compound F: accurately weighing, adding 2.5mL of physiological saline, and vortexingThe drug is completely dissolved by rotary oscillation, and the free form concentration of irinotecan in the solution is 4.0 mg/mL^-1。

3.3 animal grouping and administration

Animal groups and dosing regimens are shown in table 9. The first administration was started on the day of grouping, and the experiment was ended after 21 days, and the administration volumes were all 10 mL/kg^-1. Group 1 was a solvent control group, and the solvent was administered to the tail vein by injection 1 time every 4 days for 3 times (Q4D × 3). In groups 2 to 5, test samples of irinotecan, nktr-102 and the test compound were administered by tail vein injection at a dose of 40 mg/kg^-1(calculated as irinotecan content), Q4D × 3.

TABLE 9 drug effect experiment dosing regimen for nude mouse transplantable tumor model

4. Data recording, calculation formula

Animal survival time was recorded.

5. Statistical analysis method

Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. The two groups were compared using the t-test.

6. Results

See Table 10

TABLE 10 animal survival time (days)

P <0.05 median survival time compared to the blank solvent, irinotecan and nktr-102 group

The experimental result shows that the compound has good inhibition effect on brain glioma and is superior to irinotecan and nktr-102.

Claims

1. A multi-branched drug conjugate having the following structural formula (i):

t is GE 11;

d is camptothecin drug shown as formula (II):

R₁-R₅independently of one another, from the following groups: hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxy, cyano, nitro, azido, amido, hydrazine, amino, substituted amino, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, alkylsulfonyloxy; r₆Is H OR OR₈，R₈Is alkyl, alkenyl, cycloalkyl, haloalkyl or hydroxyalkyl; r₇Is a hydroxyl group;

"R" is selected from:

POLY is selected from

Wherein the content of the first and second substances,

representing the junction of atoms, marks "&The oxygen atom of the number is an atom connected to the organic center "R", k is an integer in the range of 50 to 200, and R is any integer between 1 and 10.

2. The multi-branched drug conjugate of claim 1, which is represented by structural formula (iii), (iv), or (v):

3. the multi-branched drug conjugate of claim 1, wherein POLY is

4. The multi-branched drug conjugate of claim 2, or a pharmaceutically acceptable salt thereof, which is:

5. the multi-branched drug conjugate according to claim 4, wherein k has an average value of 113, or a pharmaceutically acceptable salt thereof.

6. The multi-branched drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein L is:

7. the multi-branched drug conjugate according to claim 6, wherein D is irinotecan, SN-38, 10-hydroxycamptothecin, or rubitecan, or a pharmaceutically acceptable salt thereof.

8. The multi-branched drug conjugate of claim 7, or a pharmaceutically acceptable salt thereof, which is:

and salts thereof.

9. The multi-branched drug conjugate of claim 8, or a pharmaceutically acceptable salt thereof, which is:

10. a pharmaceutically acceptable composition comprising the multi-branched drug conjugate of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

11. Use of the multi-branched drug conjugate according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cancer.

12. Use of a multi-branched drug conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma and malignant sarcomas, carcinomas and lymphomas of the kidney, bile duct and brain.

13. Use of a composition according to claim 10 for the preparation of a medicament for the treatment of cancer.

14. Use of a composition according to claim 10 for the preparation of a medicament for the treatment of colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma and malignant sarcomas, carcinomas and lymphomas of the kidney, bile duct and brain.